Malarial merozoites use an array of ligands, including members of the Reticulocyte Binding Like (RBL) super-family of invasion proteins, to identify and invade erythrocytes. RBL family members are large Type I membrane anchored proteins expressed at the invasive end of merozoites that share homology with the Reticulocyte Binding Proteins 1 and 2 (PvRBP1 and 2) of Plasmodium vivax. Plasmodium species vary widely both in the number and sequence of their RBL genes, with the recently completed Plasmodium falciparum genome containing five RBL genes. Of these, three encode proteins shown to be involved in erythrocyte invasion, a fourth is a pseudogene, and the role of the fifth is as yet unclear. In order to identify sequence similarities and differences that may have functional implications for erythrocyte invasion as well as to gain insights into the recent evolutionary history of the P. falciparum RBL genes, we have sequenced all five corresponding RBL genes from the chimpanzee parasite Plasmodium reichenowi, which is the closest phylogenetic relative of P. falciparum, yet is unable to invade human erythrocytes. Two of the five P. falciparum RBL genes have highly conserved complete open reading frames in both species, while the other three genes show evidence of gene conversion and rapid evolution. The RBL super-family, therefore, appears to be surprisingly dynamic and divergent, implying that it is involved in species-specific aspects of erythrocyte recognition and invasion.

Plasmodium vivax merozoite surface protein-9 (Pvmsp-9) is characterized here along with orthologues from the related simian malarias Plasmodium cynomolgi and Plasmodium knowlesi. We show that although the corresponding MSP-9 proteins do not have acidic-basic repeated amino acid (aa) motifs, they are related to the Plasmodium falciparum acidic-basic repeat antigen (ABRA) also known as p101. Recognition of this new interspecies Plasmodium MSP family stems from the prior identification of related MSP termed PvMSP-185, PcyMSP-150, and PkMSP-110 on the surface of P. vivax, P. cynomolgi and P. knowlesi merozoites. A clone containing the nearly complete P. knowlesi gene encoding PkMSP-110/MSP-9 provided a hybridization probe and initial sequence information for the design of primers to obtain the P. vivax and P. cynomolgi orthologues using polymerase chain reaction (PCR) amplification strategies. The P. vivax, P. cynomolgi and P. knowlesi msp-9 genes encode proteins that range in calculated molecular mass from 80 to 107 kDa, have typical eukaryotic signal peptides and diverse repeated motifs present immediately upstream of their termination codon. Another feature conserved among these proteins, including the P. falciparum ABRA protein, is the positions of four cysteine residues near the N-terminus, suggesting this conservation maintains structural and perhaps functional characteristics in the MSP-9 family. Rabbit polyclonal antisera raised against recombinantly expressed N-termini of P. knowlesi and P. vivax MSP-9 cross-react with the counterpart proteins in immunofluorescence and immunoblot assays. Comparative interspecies investigations of the potential role(s) of Plasmodium MSP-9 in merozoite invasion of erythrocytes and as a malaria vaccine candidate can now be pursued.

Plasmodium knowlesi variant antigens are expressed at the surface of infected erythrocytes and are encoded by the Schizont Infected Cell Agglutination variant antigen (SICAvar) multigene family. The 3' region of the SICAvar gene locus encoding the 205 kDa variant antigen expressed in the Pk1(B+)1+ parasites was found to be altered compared to the Pk1(A+) parental clone. Here we report that this alteration is the result of a DNA rearrangement and that the original and altered 205 SICAvar alleles appear to encode bona fide variant antigens. Importantly, 205A and 205B SICAvar RNA sequences are detectable in similar apparent quantities as determined by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) amplification experiments. However, expression of the 205 kDa SICA protein at the surface of the infected erythrocyte is not characteristic of the Pk1(A+) parasites and the 205 SICAvar transcript has not been detected in Pk1(A+) parasites by northern blot analysis. Furthermore, we report that many distinct SICAvar transcripts were detected in P. knowlesi Pk1(B+)1+ cDNA library hybridization screens. Of special interest, in light of these data, distinctive differences at the 3' end of the 205A and 205B alleles are observed, which may be of functional importance. An analysis of the 3' untranslated region (UTR) of SICAvar genes in more than 100 sequences revealed a surprising common sequence pattern characterized by blocks of imperfect, GT-rich, heptad repeated motifs (Block I), followed by A and T rich homopolymers (Block II) and in a large number of genes, GC-rich segments (Block III). We show that this region undergoes extensive recombination and that the preferential stability of the 205 SICAvar transcript in Pk1(B+)1+ parasites may be associated with the presence of its specific Block III sequences. We speculate that the conserved yet polymorphic SICAvar 3'UTR sequences, and comparable regions in P. falciparum var genes, function in the stage-specific and developmentally regulated post-transcriptional gene silencing (PTGS) of variant antigen transcripts.

Members of the reticulocyte binding-like protein (RBL) family are merozoite-expressed proteins hypothesized to be essential for effective invasion of host erythrocytes. Proteins of the RBL family were first defined as merozoite invasion ligands in Plasmodium vivax, and subsequently in Plasmodium falciparum and other malaria parasite species. Comparative studies are providing insights regarding the complexity and evolution of this family and the existence of possible functionally alternative members. Here, we report the experimental and bioinformatic characterization of two new rbl genes in the simian malaria parasite species Plasmodium knowlesi. Experimental analyses confirm that a P. knowlesi gene fragment orthologous to P. vivax reticulocyte binding protein-1 (pvrbp1) represents a highly degenerated pseudogene in the H strain as well as two other P. knowlesi strains. Our data also confirm that a gene orthologous to pvrbp2 is not present in the P. knowlesi genome. However, two very diverse but related functional rbl genes are present and are reported here as P. knowlesi normocyte binding protein Xa and Xb (pknbpxa and pknbpxb). Analysis of these two rbl genes in Southern hybridizations and BLAST searches established their relationship to newly identified members of the RBL family in P. vivax and other species of simian malaria. Rabbit antisera specific for recombinant PkNBPXa and PkNBPXb confirmed expression of the prospective high molecular weight proteins and localized these proteins to the apical end of merozoites. Their precise location, as determined by immuno-electron microscopy (IEM), was found to be within the microneme organelles. Importantly, PkNBPXa and PkNBPXb are shown here to bind to host erythrocytes, and discussion is centered on the importance of these proteins in host cell invasion.